A molecular analysis of Src64 and its impact on cytoskeletal reorganization in the Drosophila embryo
Strong, Taylor Clinton
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Src-family kinases are involved in many processes related to development. As regulators of development, Src-family kinases are involved in cell motility, cell proliferation, and neo-angiogenesis. Src-family kinase activity has also been implicated in a number of cancers. Src64 in Drosophila encodes a non-receptor tyrosine kinase that is homologous to human Src family kinases. Src64 is involved in processes related to cell proliferation and apoptosis. Additionally, specific roles for Src64 have been identified in ring canal growth, dorsal closure, and salivary gland invagination. Our lab is specifically looking at the role of Src64 in microfilament ring constriction during cellularization. The Src64∆17 mutation deletes the first two exons of the gene and eliminates all but trace amounts of Src64 protein, suggesting that there is an alternate transcriptional start site that is unaffected by the Src64∆17 mutation. This alternate transcript may be responsible for the phenotypic differences observed between Src64 deficient flies and Src64∆17 flies. Semi-quantitative RT-PCR confirmed the presence of low levels of Src64 mRNA in Src64∆17 embryos. Using 5’ RACE analysis of Src64∆17 embryos we located the alternate transcriptional start site and identified a previously unknown exon located 13,100 base pairs downstream of the primary transcriptional start site, and 12,113 bp downstream of the two exons deleted in Src64∆17. To further investigate the role of Src64 in development we identified ten missense mutations in the Src64 coding region. This approach has allowed us to isolate several Src64 mutations in an unbiased manner that disrupt the SH2 and tyrosine kinase domains. We analyzed embryos affected by these Src64 mutations for defects related to Src64 activity. In our analysis we identified defects to the cellularization front and microfilament rings of varying strength depending on the specific Src64 mutation.